目的 基于Kelch样ECH相关蛋白1(Kelch-like ECH-associated protein 1,Keap1)/核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)/抗氧化反应元件(antioxidant response elements,ARE)通路和铜稳态研究珠子参总皂苷(total saponins of Panax japonicus,TSPJ)对伊利司莫和二水合氯化铜(elesclomol-CuCl₂·2H₂O,Ele-Cu²⁺)诱导的肝细胞铜死亡的影响。方法 设置对照组、模型组、TSPJ(25 μg/mL)组、Nrf2抑制剂ML385(10 μmol/L)组和TSPJ(25 μg/mL)＋ML385(10 μmol/L)组。构建Ele-Cu²⁺诱导的AML-12细胞铜死亡模型,给予药物干预后,采用MTT法检测细胞活力;采用试剂盒检测乳酸脱氢酶(lactate dehydrogenase,LDH)释放率及细胞中过氧化氢酶(catalase,CAT)、谷胱甘肽(glutathione,GSH)、丙二醛(malondialdehyde,MDA)、髓过氧化物酶(myeloperoxidase,MPO)、超氧化物歧化酶(superoxide dismutase,SOD)、总抗氧化能力(total antioxidant capacity,T-AOC)水平;采用Sytox Green、DCFH-DA、JC-1、R6G荧光探针分别检测细胞坏死性凋亡、活性氧(reactive oxygen species,ROS)水平、线粒体膜电位(mitochondrial membrane potential,MMP)和Cu²⁺水平;采用qRT-PCR和Western blotting检测细胞中Keap1、Nrf2、谷氨酸半胱氨酸连接酶催化亚基(glutamate-cysteine ligase catalytic subunit,GCLC)、血红素加氧酶-1(heme oxygenase-1,HO-1)、NAD(P)H醌氧化还原酶1[NAD(P)H quinone oxidoreductase 1,NQO1]、二氢硫辛酸乙酰转移酶(dihydrolipoamide acetyltransferase,DLAT)、铁氧还蛋白1(ferredoxin 1,FDX1)、硫辛酸合酶(lipoic acid synthase,LIAS)、热休克蛋白70(heat shock protein 70,HSP70)、三磷酸腺苷(adenosine triphosphate,ATP)酶铜转运蛋白7A(ATPase copper-transporting 7α,ATP7A)、ATP酶铜转运蛋白7B(ATPase copper-transporting 7β,ATP7B)、铜离子通道溶质载体家族31成员1(copper ion channels solute carrier family 31 member 1,SLC31A1)、抗氧化剂1铜伴侣蛋白(antioxidant 1,ATOX1)、超氧化物歧化酶的铜伴侣蛋白(copper chaperone for Sod1,CCS)、细胞色素C氧化酶铜伴侣蛋白17(cytochrome C oxidase 17,COX17)mRNA和蛋白表达。结果 与模型组比较,TSPJ组细胞存活率和MMP水平显著升高(P＜0.05、0.01),细胞坏死性凋亡率、LDH释放率、ROS、Cu²⁺、MDA水平和MPO活性显著降低(P＜0.05、0.01),CAT、GSH、SOD、T-AOC活性显著升高(P＜0.01),Nrf2、GCLC、HO-1、NQO1、FDX1、LIAS、DLAT、ATP7A、ATP7B、CCS、COX17 mRNA和蛋白表达水平显著升高(P＜0.01),Keap1、HSP90、SLC31A1、ATOX1 mRNA和蛋白表达水平显著降低(P＜0.01);而ML385能够显著抑制TSPJ对细胞铜死亡的改善作用(P＜0.01)。结论 TSPJ可能通过激活Keap1/Nrf2/ARE信号通路,调节铜稳态改善Ele-Cu²⁺诱导的肝细胞铜死亡。

Objective To investigate the effect of total saponins of Panax japonicus (TSPJ) on cuproptosis induced by elesclomol-CuCl₂·2H₂O (Ele-Cu²⁺) in hepatocytes based on Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response elements (ARE) pathway and copper homeostasis. Methods Control group, model group, TSPJ (25 μg/mL) group, Nrf2 inhibitor ML385 (10 μmol/L) group and TSPJ (25 μg/mL) + ML385 (10 μmol/L) group were set up. Ele-Cu²⁺-induced cuproptosis model in AML-12 cells was established. After drug intervention, cell viability was detected by MTT; The release rate of lactate dehydrogenase (LDH), as well as levels of catalase (CAT), glutathione (GSH), malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in cells were detected by reagent kits; The levels of necrotic apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and Cu²⁺ were respectively detected by Sytox Green, DCFH-DA, JC-1 and R6G fluorescent probes; The mRNA and protein expressions of Keap1, Nrf2, glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), dihydrolipoamide acetyltransferase (DLAT), ferredoxin 1 (FDX1), lipoic acid synthase (LIAS), heat shock protein 70 (HSP70), ATPase copper-transporting 7α (ATP7A), ATPase copper-transporting 7β (ATP7B), copper ion channels solute carrier family 31 member 1 (SLC31A1), antioxidant 1 (ATOX1), copper chaperone for Sod1 (CCS) and cytochrome C oxidase 17 (COX17) were detected by qRT-PCR and Western blotting. Results Compared with model group, survival rate and MMP level in TSPJ group were significantly increased (P < 0.05, 0.01), the necrotic apoptosis rate, LDH release rate, levels of ROS, Cu²⁺, MDA and activity of MPO were significantly reduced (P < 0.05, 0.01), the levels of CAT, GSH, SOD and T-AOC were significantly increased (P < 0.01), the mRNA and protein expressions of Nrf2, GCLC, HO-1, NQO1, FDX1, LIAS, DLAT, ATP7A, ATP7B, CCS, COX17 were significantly up-regulated (P < 0.01), the mRNA and protein expressions of Keap1, HSP70, SLC31A1, ATOX1 were significantly down-regulated (P < 0.01). While ML385 could significantly inhibit the amelioration effect of TSPJ on cell cuproptosis (P < 0.01). Conclusion TSPJ may ameliorate Ele-Cu²⁺-induced cuproptosis in hepatocytes by activating Keap1/Nrf2/ARE pathway and regulating copper homeostasis.

R285.5

湖北省科技厅重点研发项目（2025BCB067）；湖北省科技厅重点研发大健康计划项目（2022BCE017）；湖北省科技厅自然科学基金项目（2022CFB357，2022CFB427，2023AFB600）；湖北省卫生健康委员会中医药重点项目（ZY2023Z015）；湖北卫生健康委员会卫生健康科研项目（WJ2023M153）；湖北省功能性消化系统疾病中医临床医学研究中心开放基金项目（SXZ202303，SXZ202308，SXZ202311）；湖北省宜昌市科学技术局医疗卫生研究项目（A23-1-061）；肿瘤微环境与免疫治疗湖北省重点实验室2022年度开放基金（2022KZL2-11）